1. Field of the Invention
The invention relates to a recombinant cyanobacterium comprising an oxygen-tolerant, hydrogen-evolving hydrogenase, and methods of use.
2. Background Information
Cyanobacteria include a large group of photoautotrophic microorganisms. Both cyanobacteria and green algae have attracted considerable attention since they can split water photolytically to produce H2, a clean alternative to gasoline and other fossil fuels. However, one major drawback of this process is that their H2-evolving hydrogenases are extremely sensitive to O2. Thus, to realize its promising potential, a novel O2-tolerant photo-biological system needs to be developed. Transferring O2-tolerant NiFe-hydrogenase into these microbes is one of the approaches currently used to overcome the O2 sensitivity issue. Searching for new O2-tolerant hydrogenases will thus facilitate constructing such photo-biological systems.
The oceans harbor an abundance of microorganisms with H2-production capability, in particular photosynthetic bacteria. Thus far, many of these microorganisms are not identified and their functions remain unknown. These marine microbes are good resources for searching for new genes, such as novel O2-tolerant hydrogenase genes. The J. Craig Venter Institute has an ongoing global ocean microbial sampling expedition, which explores marine bacteria in a culture-independent manner by isolating DNA from ocean samples and transforming it into DNA clones for whole-genome shotgun sequencing. A pilot project for this expedition, conducted in the Sargasso Sea off Bermuda, resulted in the discovery of a total of 1.045 billion base pairs of nonredundant sequences, which are estimated to derive from 1800 genomic species, including 148 previously unknown bacterial phylotypes (Science, 304: 66-74, 2004). To take advantage of the environmental genetic information generated in this project, we searched the Sargasso Sea databases for putative NiFe-hydrogenases by using probabilistic modeling approaches such as Hidden Markov Models (HMMs).
Because a large array of genetic techniques are available for cyanobacteria, and their photosystems and H2 evolution systems are well studied, they are attractive candidates for conversion of solar energy into H2. However, nearly all naturally occurring hydrogenases are inhibited by oxygen, which leads to discontinuity of H2 photo-production. Accordingly, there remains a need for a microorganism capable of using solar energy to split H2O into H2 and O2 in a process that can be carried out in the presence of oxygen.